This application is accompanied by a Sequence Listing for nucleotide sequences.
The present invention relates to an animal feed comprised of an amount of cereal grain and an amount of polypeptide, with such polypeptide expressed by a transformed organism. More particularly, the present invention relates to a genetically engineered yeast cell that expresses a polypeptide that is comprised of amino acids necessary to animal nutrition and growth, and methods related thereto.
Cereal grains, such as corn, wheat, and rice, are fed to livestock and other animals, with consumption of these grains causing the animals to gain weight. Generally, consumption of such grains and/or grass results in sufficient weight gain, however, it is preferred to produce animals which have enhanced weight gain, as this increases the value of the animal. In other words, it is desired to produce animals of exceptional size and weight, which are comparatively larger than animals fed a normal diet. Increased weight gain can be promoted by ensuring that the animal receives necessary amino acids in desired amounts which help promote weight gain and are important nutritional constituents. It should also be noted that many amino acids which help enhance weight gain in various animals are not readily found in grass and cereal grains typically grains typically consumed by such animals. As such, farmers, ranchers, and feed lots supplement the animal""s diet with various compositions which contain many of the desired amino acids.
One way to ensure that an animal""s diet is supplemented with amino acids in amounts sufficient to result in weight gain, is to add amino acids derived from various natural sources to the animals"" feed. Natural amino acid sources include offal from the slaughter of animals for human consumption, slaughter house sludge, poultry byproduct meal, or livestock or poultry litter. An example of how this material is used to promote growth can be illustrated by the use of the sludge. The sludge, which includes blood and feces from the floor of a slaughter house is gathered, treated, and mixed with animal feed to provide an animal with amino acids. Often, bacteria, such as E. coli, Salmonella, or other pathogens, are residents of such sludge and waste. If the sludge or waste is not thoroughly treated then infectious agents can be transmitted to animals that are fed the amino acid enhancements. The risk of transmitting infectious agents to animals is a growing concern as witnessed in Europe by the reaction to xe2x80x9cmad cows"" disease.xe2x80x9d Thus, it is greatly desired to have a method for supplying enhanced levels of amino acids to animals that does not require the use of amino acids derived from animal waste or sludge.
An alternative to using animal waste products is to produce the necessary and desired amino acids through genetic modification of various microorganisms. Currently, most amino acids used to supplement animal feed are synthesized by microbial fermentations. Such amino acids are then purified and can be added to animal feed as a supplement. Most of these amino acids form heterologous peptides. The use of these techniques has been undesired because the cost has made the addition of such amino acids prohibitive. Another problem is that the excreted amino acids must be purified. Most purification methods include stabilizing the amino acid using hydrochloride with lysine. This converts D isomers to L isomers. Consequently, it is further desired to have a method or composition for adding amino acids to animal feed that does not require a purification step and is economical and cost effective.
Purified amino acids produced by genetically modified organisms, typically bacteria, suffer from a number of other problems in addition to the cost. Often, purified amino acids are subject to degradative reactions during processing, such as maillard reactions. As such, it is hypothesized that providing the amino acids in a protein or polypeptide form would prevent or render such amino acids less susceptible to such degradative reactions. Often, pure amino acids are susceptible to incongruous absorption and catabolism by the intestinal or hepatic tissue of the animal consuming such products. For this reason, it is desired to have an amino acid or protein peptide that allows for easy digestion and absorption to be preferably accomplished congruently with other dietary proteins.
Finally, when using purified amino acids, only a limited number can be provided to the animal. As such, it is desired to have a method or composition that allows for a protein or polypeptide to provide second and third limiting amino acids.
The use of yeast and other microbes to express polypeptides is known. For example, U.S. Pat. No. 5,856,123 (""123) discloses a DNA expression vector for expressing a polypeptide. The vector includes bacterial and yeast origins of replication and genes for phenotypic selection of both bacterial and yeast moieties. The expression vector is designed for expressing A or B chains of human insulin. The patent does not disclose the use of a transformed yeast cell to produce the amino acids necessary to animal nutrition. Nowhere is an animal feed mentioned in the ""123 Patent. While this particular patent is indicative of a variety of disclosed methods and compositions which discuss the use of transformed yeast cells for producing polypeptides or proteins, it is believed that the use of transformed yeast cells to produce animal feeds has not been disclosed. It is believed that the use of transformed organisms to produce polypeptides comprised of different amino acids desired in animal nutrition has not been practiced.
Yeast has also been known to be used to produce lactic acid. Metabolic engineering has focused on the production of heterologous proteins and peptides, including producing such proteins or peptides in transformed yeast. It is desired, however, to produce polypeptides comprised of more than one amino acid residue.
For these reasons, it is desired to have a method for producing an animal feed that is economical and does not run the risk of microbial contamination. Additionally, it is desired to have an easily activated vehicle that readily delivers amino acids for animal consumption. Most importantly, it is desired to have an animal feed having desired types and concentrations of amino acids.
The present invention relates to an animal feed that includes an amount of cereal grain and a peptide or a polypeptide expressed by a transformed organism. The polypeptides can be separated from the transformed organism, or the transformed organism and polypeptide, together, can be added to the cereal grain. The present invention further relates to the transformed organism which is preferably a yeast cell having a nucleic acid polymer inserted into the yeast cell chromosome, whereby the nucleic acid molecule can be expressed to form a peptide or polypeptide useful in the nutrition of an animal. The nucleic acid polymer will preferably be comprised of a fragment for expressing the polypeptide, a promoter that can be induced, and an identifier which is preferably either histidine or uracil related. The promoter is preferably a GAP promoter. While yeast is the preferred organism to be transformed, any of a variety of other microorganisms that can be consumed by animals and which can be transformed may also be used.
The present invention also relates to a method for forming the transformed organism and a method for forming the animal feed comprised of the cereal grain and either the transformed organism or the expression product of the transformed organism. The method includes preferably forming a synthetic nucleic acid polymer and attaching a promoter and stop sequence thereto. After formation of the nucleic acid polymer, the method will further include inserting the polymer into a vector and transforming a host organism. Alternatively, the integration construct can be placed in the host organism by using electro-poration. It is preferred if prior to transformation, the nucleic acid polymer is annealed or attached to primers which will allow for the formation of an integration construct. The resultant integration construct is then placed in a transfer vector and used to transform the host cell organism. It should also be noted that it is preferred if the transformed yeast cell is haploid, because it is preferred to insert the nucleic acid polymer into the chromosome of the host. Diploid hosts can be used if the nucleic acid polymer is inserted outside the chromosome.
The polypeptide or peptide expressed by the transformed organism will be at least two amino acids long, and will be comprised of at least two different amino acid residues. More preferably, the polypeptide will be between 20 and 30 amino acids long and will be comprised of at least five different residues. The amino acids to be expressed by the transformed organism will be selected in advance to ensure that, when consumed, the animal will receive maximum nutritional benefits so as to enhance growth and weight gain. Such amino acids can be selected according to the animal""s age and particular nutritional needs.
The present invention is advantageous because it allows for an economical way to produce amino acids, specifically polypeptides, that can then be used to supplement an animal""s diet. The present invention is especially advantageous because the transformed host organism can also be fed to the animal. This means purification steps can be eliminated so that costs are minimized. The invention is further advantageous because the amino acids are readily absorbed and do not readily degrade. Also, the polypeptide will include amino acid residues not typically found in other animal feeds or supplements. Thus, an animal feed is produced that is more nutritionally complete because it contains desired amino acids believed not to be found in other animal feeds.